Polarization excitation fluorimetry is directed to the measurement of the polarization of fluorescence of a fluorescent sample. Fluorescence polarization (or, emission polarization, P) is an analytical fluorescence parameter whose value aids in the analysis of electronic structure and the molecular movements of biomolecules. Fluorescence measurements are useful in chemical and biochemical research because of their sensitivity, selectivity and precision. However, difficulties in the measurement of P present themselves using currently available devices.
Fluorescence spectrometers (or, fluorimeters) are instruments designed to collect polarization of fluorescence spectra to provide information used to calculate analytical fluorescence parameters. Emission from fluorescence samples is polarized if the excitation light is polarized. This results from the photoselection of fluorophors according to their orientation. Fluorescence spectrometers thus often contain polarization modules generally consisting of one, two or three polarizers oriented at variable positions in a light beam relative to a sample. The signal output from the polarization module is then detected by one or more detectors such as photomultipliers and the measurements used to calculate P.
Polarization of fluorescence emission can be measured by analyzing the components of emission caused by excitation of a sample with polarized light. Polarization of fluorescence, P, is defined as:                     P        =                                            I              //                        -                          I              ⊥                                                          I              //                        +                          I              ⊥                                                          Eq        .                                  ⁢        1            
Referring to FIG. 1, a typical L-format arrangement is depicted. Using this arrangement to measure P, an input polarizer 30 is oriented vertically so that a sample 40 is excited with vertically polarized light. That is, the electric vector of the excitation light beam 22 is oriented parallel to the vertical, or Z, axis.
The intensity of the emitted light beam 24 is measured through an output (analyzer) polarizer 32. When the output polarizer 32 is oriented parallel (∥) to the direction of the vertically polarized excitation beam 22, the measured intensity of light is called I∥. When the output (analyzer) polarizer 32 is oriented perpendicularly (⊥) to the vertically polarized excitation beam 22, the measured intensity of light is called I⊥. Thus, under steady excitation, the output (analyzer) polarizer 32 is alternately rotated between vertical and horizontal to obtain measures for the two components of the emission intensity, I∥ and I⊥, respectively. With I∥ and I⊥ measurements, one can use equation 1 to calculate the fluorescence polarization, P.
Anisotropy (r) may also be calculated using the equation:                     r        =                                            I              //                        -                          I              ⊥                                                          I              //                        +                          2              ⁢                              I                ⊥                                                                        Eq        .                                  ⁢        2            
Anisotropy (r) is the character of a substance for which a physical property, such as index of refraction, varies in value with the direction in or along which the measurement is made.
An important disadvantage of the apparatus and method according to FIG. 1, is that because the output polarizer 32 must be physically rotated at each wavelength at which P is to be determined in order to obtain the two intensity measurements (I⊥ and I∥) required by equations 1 and 2, it is not readily compatible with the continuous measurement of a spectrum. Mechanical rotation of the output polarizer 32 (either manual or automated using a stepping motor or the like) may be employed, but this leads to limited time resolution. It also leads to a dependency upon the polarization sensitivities of other analyzing components. This is because a detector 60 “sees” two different polarization states when measuring I⊥ and I∥. The detector 60 used to detect the output signal 26 measures the intensity of light but is affected by the polarization too. When changes to the polarization state occur as a result of movement of the polarizer 32, the detector 60 responds inappropriately and the resulting measurements require correction. Because it is highly likely that the response of the detector 60 will change with polarization, the determination of P is thus compromised. Errors in the determination of I∥ and I⊥ are likely because the sensitivity of the detector 60 to vertically or horizontally polarized light is different.
Another method used to measure P is the T-format or two-channel method shown schematically in FIGS. 2a and 2b. In this second method, two similar detectors 60 are used to measure the parallel and perpendicular components (I∥ and I⊥) simultaneously. When two emission polarizers 32 are employed, they do not need to move, so that mechanical considerations do not apply.
However, this technique demands that the relative sensitivity of the two detectors be determined and used to correct the measurement. Special measurements must be made to find the relative sensitivity of the two detectors 60 (i.e., G, or the relationship between the two detectors). This requires that the excitation polarizer 30 be physically placed in the horizontal position (FIG. 2a) and then manually rotated to the vertical position (FIG. 2b) to complete the standardization.
The above-described problems exist in many currently available polarization modules of fluorescence spectrometers because of their employment of one or two polarizers that are not fixed in position relative to the sample (as depicted in FIG. 1 or 2). As discussed above, the required mechanical movement, or rotation, of the polarizer(s) slows the rate of operation. In addition, the movement of the polarizer(s) results in changes to the polarization state of the light seen by the detector and these changes require corrections, further measurements or both.
Considering again the L-format (single detector) method, the above problems might be addressed by fixing the output (analyzer) polarizer in the horizontal position and periodically interposing a half-wave retarder between the fluorescent sample and the output (analyzer) polarizer. Any polarization form can be converted to any other form by means of a suitable retarder. Since a half-wave retarder in effect rotates the plane of polarization 90° (recall that when the angle between the fast axis of a half-wave retarder and the input plane of polarization is 45°, vertically polarized light is converted to horizontally polarized light), the I⊥ would be measured without the plate in position and the I∥ with it in position. However, this method would be impractical because of the wavelength dependence of half-wave plates. Also, it would require mechanical components if spectra were to be automatically scanned, leading to inevitable mechanical problems.
Information relevant to attempts to address these problems can be found in U.S. Pat. No. 4,203,670 to Bromberg; U.S. Pat. No. 4,269,511 to Erwin; U.S. Pat. No. 4,516,856 to Popelka; and, U.S. Pat. No. 4,699,512 to Koshi. However, each one of these references suffers from one or more of the following disadvantages: (1) they employ one or two moving polarizers that, because of their very movement, cause noise and distortion of the signal levels detected by a detector that require corrections and further measurements to be made, and thus slow operation; (2) they utilize only a single fixed polarizer, and therefore lack a polarizer in either the input/excitation or output/emission beam; (3) they require two detectors which necessitates calibration and/or standardization (matching); and, (4) they use variable retardance modulators such as liquid crystal variable retardance modulators (LCVRs) to enable slower speed measurements, but position them in the input/excitation beam rather than in the output/emission beam and use them in combination with only a single fixed polarizer or with moving polarizer(s), thus resulting in many of the problems described above.
For the foregoing reasons, there is a need for a device and method for measuring fluorescence polarization with a polarization module that combines (1) two polarizers that are fixed in position (and thereby polarization characteristics of other components are rendered irrelevant) and bracket a sample so as to enable accurate and direct measurements at a known polarization state and to eliminate the need for correction and multiple measurements of an output signal with (2) a variable retardance modulator (i.e., variable retardance waveplate) to enable switching between two adjustable and selectable states at arbitrarily low rates for the collection and processing of fluorescence polarization (P) information at slow speeds with high resolution; and, (3) a single detector, to eliminate the need to match multiple detectors and provide lower cost.